The prevalence of chronic inflammatory and thrombotic diseases is rising with the general increase in population longevity. Our application addresses the role of neutrophil extracellular traps (NETs), chromatin released by activated neutrophils, in such age-related diseases. We hypothesize that aging promotes NET formation and NETs in turn escalate chronic inflammatory and thrombotic diseases. We propose that genes regulating NETosis, such as peptidylarginine deiminase 4 (PAD4), impact aging. Our preliminary results indicate that neutrophils from old mice make more NETs and PAD4, which orchestrates NETosis, promotes cardiac fibrosis in older mice. We observed that the mitochondrial deacetylase sirtuin 3 (SIRT3), whose regulation of reactive oxygen species (ROS) is implicated in aging, is expressed by neutrophils and platelets and is a potent down-regulator of neutrophil ROS production and NETosis. We will address our hypothesis with in vitro studies and disease models in wild type (WT) and genetically engineered mutant mice. We propose three Aims: Aim 1. To study the relationship of NETosis and aging in WT mice, the effect of calorie restriction (CR) on NETosis and resulting inflammatory, thrombotic, and fibrotic responses. We will examine NETosis and the susceptibility to cardiac and lung fibrosis as a function of mouse age. We will evaluate whether DNase1, that helps clear NETs, will reduce experimental lung fibrosis. We will study the effect of CR, known to slow down aging, on NET formation and susceptibility to thrombosis and organ fibrosis. Aim 2. To investigate whether mutations in PAD4, an enzyme required for NET formation, affect aging and age-related inflammatory and thrombotic diseases. We will study the effect of PAD4- deficiency on age-related heart fibrosis and susceptibility to experimental lung fibrosis. We will follow aging healt and longevity of PAD4-/- and WT littermates. We will genetically engineer transgenic mice overproducing PAD4 in neutrophils. We anticipate that they will produce NETs more readily and have an increased propensity towards thrombosis and interstitial lung fibrosis as well as delayed wound healing. Aim 3. To evaluate the role of SIRT3 in NETosis and in disease models involving NETs. Study the function of platelet SIRT3 in platelet activation and in their priming of neutrophils towards NETosis. We are keen to evaluate the role of SIRT3 in neutrophils, platelets and in the interaction between platelets and neutrophils that may enhance NETosis. SIRT3-/- and already generated SIRT3- overexpressing mice will be evaluated in inflammation and thrombosis. Bone marrow transplant chimeric animals will be prepared to distinguish the role of blood cell versus tissue SIRT3. We hope to uncover new pharmacological targets to reduce NETs' contribution to chronic inflammatory and thrombotic processes and that this may eventually improve the health of our aging population.